Novel human kinase and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application claims the benefit of U.S. ProvisionalApplication No. 60/297,856 which was filed on Jun. 13, 2001 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of novel human polynucleotides encoding a protein thatshares sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, andcosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

Kinases mediate phosphorylation of a wide variety of proteins andcompounds in the cell. Along with phosphatases, kinases are involved ina range of regulatory pathways. Given their physiological importance,kinases have been subject to intense scrutiny and are proven drugtargets.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of nucleotides that encode a novel human protein, andthe corresponding amino acid sequence of this protein. The novel humankinase (NHK) described for the first time herein shares structuralsimilarity with animal kinases, including, but not limited to,serine/threonine kinases, calcium/calmodulin dependent kinases, andrho-associated kinases. As such, the novel polynucleotides encode a newkinase protein having homologues and orthologs across a range of phylaand species.

The novel human polynucleotides described herein encode an open readingframe (ORF) encoding a protein of 359 amino acids in length (see SEQ IDNO: 2).

The invention also encompasses agonists and antagonists of the describedNHKs, including small molecules, large molecules, mutant NHKs, orportions thereof, that compete with native NHK, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHKs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHKs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHK sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHK. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHKs. When the unique NHKsequences described in SEQ ID NOS:1-3 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHK sequences described in SEQ IDNOS:1-3 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins which would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses.

Additionally, the unique NHK sequences described in SEQ ID NOS:1-3 areuseful for the identification of protein coding sequence and mapping aunique gene to a particular chromosome. These sequences identify actual,biologically verified, and therefore relevant, exon splice junctions asopposed to those that may have been bioinformatically predicted fromgenomic sequence alone. The sequences of the present invention are alsouseful as additional DNA markers for restriction fragment lengthpolymorphism (RFLP) analysis, and in forensic biology.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists, of NHKexpression and/or NHK activity that utilize purified preparations of thedescribed NHKs and/or NHK product, or cells expressing the same. Suchcompounds can be used as therapeutic agents for the treatment of any ofa wide variety of symptoms associated with biological disorders orimbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequence of a novel human ORF thatencodes the described novel human kinase protein. SEQ ID NO:3 describesthe NHK ORF and flanking sequences.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHK described for the first time herein, is a novel protein that isexpressed in, inter alia, human cell lines, and human lymph node, bonemarrow, esophagus, fetal kidney, fetal lung, tongue, adenocarcinoma, andosteocarcinoma cells. The described sequences were compiled from humangenomic sequence and cDNAs made from human fetal lung, fetal kidney,esophagus, and lymph node mRNAs (Edge Biosystems, Gaithersburg, Md.,Clontech, Palo Alto, Calif.).

The present invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, the expressionproducts of such nucleotides, and: (a) nucleotides that encode mammalianhomologs of the described genes, including the specifically describedNHK, and the NHK products; (b) nucleotides that encode one or moreportions of the NHK that correspond to functional domains, and thepolypeptide products specified by such nucleotide sequences, includingbut not limited to the novel regions of any active domain(s); (c)isolated nucleotides that encode mutant versions, engineered ornaturally occurring, of the described NHK in which all or a part of atleast one domain is deleted or altered, and the polypeptide productsspecified by such nucleotide sequences, including but not limited tosoluble proteins and peptides in which all or a portion of the signalsequence is deleted; (d) nucleotides that encode chimeric fusionproteins containing all or a portion of a coding region of a NHK, or oneof its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such-as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing. As discussed above,the present invention includes: (a) the human DNA sequences presented inthe Sequence Listing (and vectors comprising the same) and additionallycontemplates any nucleotide sequence encoding a contiguous NHK openreading frame (ORF) that hybridizes to a complement of a DNA sequencepresented in the Sequence Listing under highly stringent conditions,e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., NY, at p. 2.10.3) and encodes a functionallyequivalent expression product. Additionally contemplated are anynucleotide sequences that hybridize to the complement of the DNAsequence that encode and express an amino acid sequence presented in theSequence Listing under moderately stringent conditions, e.g., washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet stillencode a functionally equivalent NHK product. Functional equivalents ofa NHK include naturally occurring NHKs present in other species andmutant NHKs whether naturally occurring or engineered (by site directedmutagenesis, gene shuffling, directed evolution as described in, forexample, U.S. Pat. No. 5,837,458 or 5,723,323 both of which are hereinincorporated by reference). The invention also includes degeneratenucleic acid variants of the disclosed NHK polynucleotide sequences.

Additionally contemplated are polynucleotides encoding NHK ORFs, ortheir functional equivalents, encoded by polynucleotide sequences thatare about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NO:1 (as measured by BLAST sequence comparisonanalysis using, for example, the GCG sequence analysis package usingdefault parameters).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHK encoding polynucleotides. Such hybridization conditionscan be highly stringent or less highly stringent, as described above. Ininstances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80, or about 34 to about 45 bases long, orany variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

Alternatively, such NHK oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a micro array or high-throughput. “chip” format).Additionally, a series of the described NHK oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHK sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS: 1-3 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the sequencesof SEQ ID NOS: 1-3, or an amino acid sequence encoded thereby. Methodsfor attaching biopolymers to, or synthesizing biopolymers on, solidsupport matrices, and conducting binding studies thereon are disclosedin, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405 thedisclosures of which are herein incorporated by reference in theirentirety.

Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-3 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-3.

For example, a series of the described oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

Microarray-based analysis allows the discovery of broad patterns ofgenetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-3 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

Probes consisting of sequences first disclosed in SEQ ID NOS:1-3 canalso be used in the identification, selection and validation of novelmolecular targets for drug discovery. The use of these unique sequencespermits the direct confirmation of drug targets and recognition of drugdependent changes in gene expression that are modulated through pathwaysdistinct from the drugs intended target. These unique sequencestherefore also have utility in defining and monitoring both drug actionand toxicity.

As an example of utility, the sequences first disclosed in SEQ ID NOS:1-3 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-3 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

Thus the sequences first disclosed in SEQ ID NOS:1-3 can be used toidentify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-3. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence.

For oligonucleotide probes, highly stringent conditions may refer, e.g.,to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and60° C. (for 23-base oligos). These nucleic acid molecules may encode oract as NHK gene antisense molecules, useful, for example, in NHK generegulation and/or as antisense primers in amplification reactions of NHKgene nucleic acid sequences. With respect to NHK gene regulation, suchtechniques can be used to regulate biological functions. Further, suchsequences can be used as part of ribozyme and/or triple helix sequencesthat are also useful for NHK gene regulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine;4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupincluding, but not limited to, a phosphdrothioate, a phosphorodithioate,a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHK.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

Low stringency conditions are well-known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, NY; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, NY.

Alternatively, suitably labeled NHK nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

In another example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individual's genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

Further, a NHK gene homolog can be isolated from nucleic acid from anorganism of interest by performing. PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHK products disclosed herein. The template for thereaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue, such as prostate, rectum, colon, or adrenal gland,known or suspected to express an allele of a NHK gene.

The PQR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHK gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known to, or suspectedof, expressing a NHK gene). A reverse transcription (RT) reaction can beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with acomplementary primer. Thus, cDNA sequences upstream of the amplifiedfragment can be isolated. For a review of cloning strategies that can beused, see e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHK sequence can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown or suspected to be expressed in an individual putatively carryinga mutant NHK allele, and by extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal sequence. Using these two primers, the product is then amplifiedvia PCR, optionally cloned into a suitable vector, and subjected to DNAsequence analysis through methods well-known to those of skill in theart. By comparing the DNA sequence of the mutant NHK allele to that of acorresponding normal NHK allele, the mutation(s) responsible for theloss or alteration of function of the mutant NHK gene product can beascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry a mutant NHK allele(e.g., a person manifesting a NHK-associated phenotype such as, forexample, immune disorders, obesity, high blood pressure, etc.), or acDNA library can be constructed using RNA from a tissue known, orsuspected, to express a mutant NHK allele. A normal NHK gene, or anysuitable fragment thereof, can then be labeled and used as a probe toidentify the corresponding mutant NHK allele in such libraries. Clonescontaining mutant NHK sequences can then be purified and subjected tosequence analysis according to methods well-known to those skilled inthe art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHK allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHK product, as described below. For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.

Additionally, screening can be accomplished by screening with labeledNHK fusion proteins, such as, for example, alkaline phosphatase-NHK orNHK-alkaline phosphatase fusion proteins. In cases where a NHK mutationresults in an expression product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies toNHK are likely to cross-react with a corresponding mutant NHK expressionproduct. Library clones detected via their reaction with such labeledantibodies can be purified and subjected to sequence analysis accordingto methods well-known in the art.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos. 5,830,721and 5,837,458 which are herein incorporated by reference in theirentirety.

The invention also encompasses (a) DNA vectors that contain any of theforegoing NHK coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHK coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHK coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHK sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of a NHK,as well as compounds or nucleotide constructs that inhibit expression ofa NHK sequence (transcription factor inhibitors, antisense and ribozymemolecules, or open reading frame sequence or regulatory sequencereplacement constructs), or promote the expression of a NHK (e.g.,expression constructs in which NHK coding sequences are operativelyassociated with expression control elements such as promoters,promoter/enhancers, etc.).

Where, as in the present instance, some of the described NHK peptides orpolypeptides are likely to be cytoplasmic proteins, expression systemscan be engineered that produce soluble derivatives of a NHK(corresponding to a NHK extracellular and/or intracellular domains, ortruncated polypeptides lacking one or more hydrophobic domains) and/orNHK fusion protein products (especially NHK-Ig fusion proteins, i.e.,fusions of a NHK domain to an IgFc), NHK antibodies, and anti-idiotypicantibodies (including Fab fragments) that can be used in therapeuticapplications. Preferably, the above expression systems are engineered toallow the desired peptide or polypeptide to be recovered from theculture media.

The NHKs or NHK peptides, NHK fusion proteins, NHK nucleotide sequences,antibodies, antagonists and agonists can be useful for the detection ofmutant NHKs or inappropriately expressed NHKs for the diagnosis ofdisease. The NHK proteins or peptides, NHK fusion proteins, NHKnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHK inthe body. The use of engineered host cells and/or animals can offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to the endogenous receptor/ligand of a NHK, but canalso identify compounds that trigger NHK-mediated activities orpathways.

Finally, the NHK products can be used as therapeutics. For example,soluble derivatives such as NHK peptides/domains corresponding to theNHKs, NHK fusion protein products (especially NHK-Ig fusion proteins,i.e., fusions of a NHK, or a domain of a NHK, to an IgFc), NHKantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHK-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHK, or a NHK-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the NHK could activateor effectively antagonize the endogenous NHK or a protein interactivetherewith. Nucleotide constructs encoding such NHK products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHK, a NHK peptide, or a NHK fusionprotein to the body. Nucleotide constructs encoding functional NHKs,mutant NHKs, as well as antisense and ribozyme molecules can also beused in “gene therapy” approaches for the modulation of NHK expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 The NHK Sequences

The cDNA sequences and the corresponding deduced amino acid sequence ofthe described NHK are presented in the Sequence Listing. The NHKnucleotide sequences were obtained from cDNAs obtained using probesand/or primers generated from human genomic sequence.

Expression analysis has provided evidence that the described NHKs areexpressed in a variety of tissues, and that the NHK shares significantsimilarity with a variety of protein kinases. Given the physiologicalimportance of protein kinases, they have been subject to intensescrutiny as exemplified and discussed in U.S. Pat. Nos. 5,756,289,5,817,479 and 6,340,583, which are herein incorporated by reference intheir entirety. These issued U.S. patents additionally describe avariety of uses and applications for the described NHK.

The described NHK is apparently encoded by several exons dispersed onhuman chromosome 19 (see GENBANK accession no. AC008735). Accordingly,the described NHK can be used to map the coding region of thecorresponding human genomic locus (i.e., chromosome mapping), and toidentify exon splice junctions.

The described novel human polynucleotide sequences can be used, amongother things, in the molecular mutagenesis/evolution of proteins thatare at least partially encoded by the described novel sequences using,for example, polynucleotide shuffling or related methodologies. Suchapproaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458 whichare herein incorporated by reference in their entirety.

NHK gene products can also be expressed in transgenic animals. Animalsof any species, including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate NHK transgenic animals.

Any technique known in the art may be used to introduce a NHK transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to pronuclear microinjection(Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191);retrovirus-mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

The present invention provides for transgenic animals that carry the NHKtransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

When it is desired that a NHK transgene be integrated into thechromosomal site of the endogenous NHK gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHKgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHK gene (i.e.,“knockout” animals).

The transgene can also be selectively introduced into a particularcell-type, thus inactivating the endogenous NHK gene in only thatcell-type, by following, for example, the teaching of Gu et al., 1994,Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

Once transgenic animals have been generated, the expression of therecombinant NHK gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHK gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHKtransgene product.

The present invention also provides for “knock-in” animals. Knock-inanimals are those in which a polynucleotide sequence (i.e., a gene or acDNA) that the animal does not naturally have in its genome is insertedin such a way that the sequence is expressed. Examples include, but arenot limited to, a human gene or cDNA used to replace its murine orthologin the mouse, a murine cDNA used to replace the murine gene in themouse, and a human gene or cDNA or murine cDNA that is tagged with areporter construct used to replace the murine ortholog or gene in themouse. Such replacements can occur at the locus of the murine orthologor gene, or at another specific site. Such knock-in animals are usefulfor the in vivo study, testing and validation of, intra alia, human drugtargets, as well as for compounds that are directed at the same andtherapeutic proteins.

5.2 NHK and NHK Polypeptides

NHK products, polypeptides, peptide fragments, mutated, truncated, ordeleted forms of the NHKs, and/or NHK fusion proteins can be preparedfor a variety of uses. These uses include, but are not limited to thegeneration of antibodies, as reagents in diagnostic assays, theidentification of other cellular gene products related to the NHK, asreagents in assays for screening for compounds that can be used aspharmaceutical reagents for the therapeutic treatment of mental,biological, or medical disorders and disease.

The Sequence Listing discloses the amino acid sequence encoded by thedescribed NHK-encoding polynucleotides. The NHK has an initiatormethionine in a DNA sequence context consistent with eucaryotictranslation initiation site and lacks a signal-like sequence indicatingthat the NHK can be cytoplasmic, nuclear, secreted, or membraneassociated.

The NHK amino acid sequence of the invention include the amino acidsequence presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHK homologues from otherspecies are encompassed by the invention. In fact, any NHK proteinencoded by the NHK nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al. eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalentto the NHK products encoded by the presently described nucleotidesequences as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHK substrate, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHKproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHKnucleotide sequences described above, but which result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions can be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged, (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHK nucleotide sequences of the invention. Where the NHK peptide orpolypeptide can exist, or has been engineered to exist, as a soluble orsecreted molecule, the soluble NHK peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express a NHK, or functional equivalent, insitu. Purification or enrichment of a NHK from such expression systemscan be accomplished using appropriate detergents and lipid micelles andmethods well-known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of theNHK, but to assess biological activity, e.g., in certain drug screeningassays.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHK nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing NHK nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHK nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHK nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHK nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHKproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHK, or for raising antibodies to a NHK,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHK coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign polynucleotide sequences.The virus grows in Spodoptera frugiperda cells. A NHK coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofNHK coding sequence will result in inactivation of the polyhedrin geneand production, of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted sequence is expressed (e.g.., see Smith et-al.,1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the NHK nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric sequence may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing a NHK product in infected hosts (e.g.,See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).Specific initiation signals may also be required for efficienttranslation of inserted NHK nucleotide sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire NHK gene or cDNA, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of a NHK coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (See Bitter et. al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications'(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms' for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe NHK sequences described above can be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the NHKproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the NHK product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺-nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

Also encompassed by the present invention are fusion proteins thatdirect a NHK to a target organ and/or facilitate transport across themembrane into the cytosol. Conjugation of NHKs to antibody molecules ortheir Fab fragments could be used to target cells bearing a particularepitope. Attaching an appropriate signal sequence to a NHK would alsotransport a NHK to a desired location within the cell. Alternativelytargeting of a NHK or its nucleic acid sequence might be achieved usingliposome or lipid complex based delivery systems. Such technologies aredescribed in “Liposomes: A Practical Approach”, New, R.R.C., ed., OxfordUniversity Press, NY, and in U.S. Pat. Nos. 4,594,595, 5,459,127,5,948,767 and 6,110,490 and their respective disclosures, which areherein incorporated by reference in their entirety. Additionallyembodied are novel protein constructs engineered in such a way that theyfacilitate transport of NHKs to a target site or desired organ, wherethey cross the cell membrane and/or the nucleus where the NHKs can exerttheir functional activity. This goal may be achieved by coupling of aNHK to a cytokine or other ligand that provides targeting specificity,and/or to a protein transducing domain (see generally U.S. ProvisionalPatent Application Ser. Nos. 60/111,701 and 60/056,713, both of whichare herein incorporated by reference, for examples of such transducingsequences), to facilitate passage across cellular membranes, and canoptionally be engineered to include nuclear localization signals.

Additionally contemplated are oligopeptides that are modeled on an aminoacid sequence first described in the Sequence Listing. Such NHKoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHK oligopeptidescan be of any length disclosed within the above ranges And can initiateat any amino acid position represented in the Sequence Listing.

The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components which naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially isolated orpure protein or polypeptide may be obtained, for example, by extractionfrom a natural source, by expression of a recombinant nucleic acidencoding the protein or polypeptide or by chemically synthesizing theprotein or polypeptide.

Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants which accompany it inits natural state. Thus, a polypeptide which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

5.3 Antibodies to NHK Products

Antibodies that specifically recognize one or more epitopes of a NHK, orepitopes of conserved variants of a NHK, or peptide fragments of a NHKare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention can be used, for example, in thedetection of NHK in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHK. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHK expression product. Additionally, such antibodies can be-usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHK-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHK activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the NHK, an NHK peptide (e.g., one corresponding to afunctional domain of an NHK), truncated NHK polypeptides (NHK in whichone or more domains have been deleted), functional equivalents of theNHK or mutated variant of the NHK. Such host animals may include but arenot limited to pigs, rabbits, mice, goats, and rats, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including, but not limited to, Freund'sadjuvant (complete and incomplete), mineral salts such as aluminumhydroxide or aluminum phosphate, chitosan, surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, and potentially useful human adjuvants such as BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Alternatively, the immuneresponse could be enhanced by combination and or coupling with moleculessuch as keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid,ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies areheterogeneous populations of antibody molecules derived from the sera ofthe immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 5,877,397;6,075,181 and 6,150,584 and their respective disclosures which areherein incorporated by reference in their entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 341:544-546) can be adapted to produce single chainantibodies against NHK expression products.

Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to a NHK can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a given NHK, using techniques well-known tothose skilled in the art. (See, e.g . . . , Greenspan & Bona, 1993,FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to a NHK domain andcompetitively inhibit the binding of NHK to its cognate receptor/ligandcan be used to generate anti-idiotypes that “mimic” the NHK and,therefore, bind, activate, or neutralize a NHK, NHK receptor, or NHKligand. Such anti-idiotypic antibodies or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens involving aNHK-mediated pathway.

Additionally given the high degree of relatedness of mammalian NHKs, thepresently described knock-out mice (having never seen NHK, and thusnever been tolerized to NHK) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHK (i.e., NHK will be immunogenic in NHK knock-outanimals).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1-12. (cancelled)
 13. A substantially isolated polypeptide having theamino acid sequence of SEQ ID NO:
 2. 14. A substantially isolatedantibody having immunospecificity for the polypeptide sequence of SEQ IDNO:
 2. 15. A method of expressing the amino acid sequence of SEQ ID NO:2in a cell, comprising providing an expression vector encoding amino acidsequence of SEQ ID NO:2; introducing the vector into a cell, andmaintaining the cell under conditions permitting expression of the aminoacid sequence of SEQ ID NO:2 by the cell.